Cell Viability
Cellular energy metabolism is a complex process that allows cells to generate energy through a series of enzymatic and chemical reactions. In eukaryotes, metabolism is centered at the mitochondria and involves multiple pathways including glycolysis and oxidative phosphorylation. Depending on cell status and adaptation to various stimuli, a particular metabolic pathway is activated, and the study of the enzymatic reactions and metabolites of these pathways help researchers to understand the metabolic status of the cell as well as cell viability and toxicity. One essential aspect of cellular energy metabolism is the reduction-oxidation (redox) state of the cell. During cellular energy metabolism, energy is often stored and released as part of redox reactions. Major co-factors in these metabolic reactions are the nucleotides NAD(P) and NAD(P)H. The redox state of a cell is described as the balance between the oxidized form of these nucleotides (NAD(P)) and the reduced form (NAD(P)H). The redox state is studied in order to determine the metabolic status of live cells and can be used to study enzymes and metabolites that are involved in the redox state and/or utilized directly or indirectly the NAD(P)/NAD(P)H nucleotides. Methods currently exist to study the redox state of cells, including tetrazolium salts (MTT, MTS, and XTT) and resazurin. All of these methods involve compounds that are reduced in metabolically active cells to produce either a colorimetric or fluorescent signal. These methods are limited by low sensitivity and often require labor-intensive nucleotide extraction methods and/or enzymatic cycling reactions to amplify the signal. These molecules vary structurally, and it is not known what cellular enzymes reduce each compound; consequently, the design of new molecules to study the redox state is not obvious.
NAD(P)/NAD(P)H
The molecules NAD and NADP, and their reduced forms NADH and NADPH, are cofactors present in all organisms. They are involved in many and multiple oxidoreductase reactions critical to cell metabolism as well as function in other necessary cellular processes. Often, it is desirable to measure the levels of NAD, NADP, NADH, NADPH as an indication of cellular redox state and its perturbation by treatments.